Spectroscopy is a method for obtaining information on a molecular scale by the use of light. This information can be related to the rotational, vibrational and/or electronic states of the molecules probed as well as dissociation energy and more. The rotational and/or vibrational spectrum of a given molecule is specific for that molecule. As a consequence, molecular spectra are often referred to as ‘fingerprints’ related to a specific molecule. Information related to in particular rotational, vibrational and/or electronic states of molecules can therefore be used to analyze a sample comprising a number of unknown molecular components, thereby obtaining knowledge about the molecular components in the sample.
The basis for a spectroscopic setup is a light source, e.g. a laser, which is used for illuminating a sample. The light from the light source (the incoming light) will interact with the sample, which often results in an alternation of the light which is transmitted through, emitted by, reflected by and/or scattered by the sample. By collecting the altered light and analyzing its spectral distribution, information about the interaction between the incoming light and the molecular sample can be obtained; hence information about the molecular components can be obtained.
The spectral distribution is typically measured by using a spectrometer. A spectrometer is an optical apparatus that works by separating the light beam directed into the optical apparatus into different frequency components and subsequently measures the intensity of these components by using a CCD detector, a CCD array, photodiode or such.
The altered light reflecting interactions between the incoming light and the molecular sample can normally be characterized as either emission or scattering. Emission signals have relatively broad spectral profiles as compared to scattering light signals, which normally display quite narrow spectral lines. One process is often dominating over the other, but both processes can and will most often occur simultaneously. The intensity of the emitted light vs. the intensity of the scattered light depends among others on the frequency and the power of the incoming light, the intensity of the incoming light at the measuring point in the sample, and the molecular components in the sample.
Emission describes the process when a molecule absorbs light from a light source, e.g. a laser, and afterwards emits light again. The emitted light is normally characterized by having a different spectral distribution compared to the incoming light, and will have a relatively broad spectral distribution reflecting the different rotational and/or vibrational states of the electronic state(s) in the molecules. The majority of emission processes can be characterized as either fluorescence or phosphorescence, where the spin of the electronic states in the molecule involved in absorption and emission of light is the same in the fluorescence process but different in the phosphorescence process. In general, fluorescence can be characterized as a spectroscopically allowed process, whereas phosphorescence is a spectroscopically forbidden process based on the conversion and the alteration of the electronic state spins, respectively. The intensity of phosphorescence signals is consequently normally much weaker than fluorescence signals.
Scattered light can be classified as being either elastic or inelastic and is characterized by being spectroscopically very narrow signals. Elastic scattering is referred to as Rayleigh scattering, in which there is no frequency shift, i.e. Rayleigh scattering has the same frequency as that of the incoming light.
The most commonly known example of inelastic scattering is Raman scattering, in which there is an energy interchanging between the molecule and the photons of the incoming light. The frequencies, i.e. the spectral distribution of the Raman scattered light, will be different from that of the incoming light and uniquely reflect the specific vibrational levels of the molecule; hence it is a fingerprint spectrum. This can be used for identification of the molecular composition of the substance probed and/or the concentration of the specific molecules in the substance.
Raman scattering is a relatively weak process compared to Rayleigh scattering and fluorescence. Reduction of contributions from these other processes is thus desirable when collecting Raman scattered light. In addition, the intensity of the Raman scattered light depends strongly on the frequency and the intensity of the incoming light. It is therefore essential to monitor power fluctuations in the incoming light if one is to receive reliable information about the distribution of molecular components in different samples and/or sample spots based on an analysis of the collected Raman scattered light. The same is true if the analysis of the molecular components in a sample and/or different sample spots is based on emission spectra.
In order to collect the altered light and direct it into an apparatus, e.g. a spectrometer, for the subsequent analysis, an optical probe is required. Such normally comprises a combination of different optical components, like lenses, mirrors and fibers, and is characterized by having a leg for the incoming light and a leg for the altered light.
A microscope can be used as an optical probe or incorporated as part of one. A microscope objective in the microscope focuses the incoming light onto a sample and collects the altered light. Alternatively, a second microscope objective can be employed for collecting the altered light. A microscope-based optical probe is not a movable object, and the samples studied with such a probe consequently need to be inserted into the microscope or placed on top of it depending on the direction of the incoming light and the position of the microscope objective. Samples collected in vitro and placed on e.g. cover slips or other types of thin plates are preferable and easy to work with in a microscope. Measurements of e.g. blood sugar levels in a patient can be performed when provided with a blood sample from the patient. However, it requires an educated person to obtain a blood sample from a patient and the process if self can be somewhat unpleasant for the patient. An alternative to this in vitro method is for the patient to insert his/her arm directly under or above the microscope objective in the microscope for an in vivo measurement of the blood sugar level. Unfortunately, this is cumbersome if not impossible with most microscopes.
An optical probe employing not the entire microscope but only microscope objective(s) mounted separately on e.g. a table allows for a larger accessibility between probe and sample. In vivo measurements of blood sugar levels in a patient become more convenient as the patient's arm or finger can be placed in front of the microscope objective(s) without much difficulty. However, if the chosen sample is a leg, it might prove more difficult to place it appropriately in front of the microscope objective(s). Furthermore, in vivo diagnostics of skin abnormalities in the cervix, i.e. examination of the potential risk of cervical cancer, are impossible to perform using a microscope objective mounted on a table or such.
Consequently, there is a need for a movable and flexible optical probe in order to measure optical signals in vivo. One way to solve this is to employ fibers for guiding the light into and/or away from the probe. Different examples of such can be found in the literature.
The optical probe described in U.S. Pat. No. 5,842,995 finds its primary application within the field of diagnostic of skin abnormalities as a result of e.g. cancer, and is based on fibers both for directing the incoming light onto the sample and for collecting the altered light from the sample. The incoming light passes through a broadband filter before it reaches the sample and the altered light from the sample is collected in a multicore fiber. The leg for the incoming light and the leg for collecting the altered light are aligned co-parallelly and share no optical components.
The optical probe found in U.S. Pat. No. 5,112,227 comprises a leg for the incoming light and a leg for collecting the altered light, where the two legs are aligned co-parallelly, and share the same lens for focusing the light into the sample and for collecting the altered light from the sample. An optical filter placed at a 45° angle before the lens, allows the incoming light to pass through and reflects the altered light from the sample, thereby separating the two optical legs.
In Journal of Biomedical Optics vol. 8, page 221-147 (2003) (referred to as J. Bio. Opt. from hereon), several different optical probes are described. The probes are primarily multi-core fiber probes without optical focusing means. The majority of the probes have a leg for the incoming light and a leg for collecting the altered light, where the two legs are aligned co-parallelly. U.S. Pat. No. 5,842,995, U.S. Pat. No. 5,112,227, and J. Bio. Opt. all describe flexible and movable optical probes. However, none of these probes account accurately for intensity variations in the incoming light.
The process of coupling laser light into a fiber is quite sensitive to the angle at which the laser light is focused into the fiber and the distance between the focus point of the lens, which focuses the laser into the fiber, and the fiber itself. Variations in the intensity of light coming out a fiber will vary as a result of the efficiency by which the laser light is coupled into the fiber. As a consequence, alternations in the intensity of the altered light from the sample will both reflect intensity variations in the incoming light and variations within the sample. Means for accurate detection of the intensity of the incoming light directly before the light is focused into the sample, are thus crucial if one wants to obtain an intensity variation pattern solely reflecting sample variations. Common for the optical probes described in U.S. Pat. No. 5,842,995, U.S. Pat. No. 5,112,227, and J. Bio. Opt., is that none of them provide this.
In addition, when the incoming light is focused into the sample, the altered light will not only come from the focus spot of the incoming light, but also from the cone-shaped area both above and below the focus spot. Hence, the light signals measurable with the probes described in U.S. Pat. No. 5,842,995, U.S. Pat. No. 5,112,227, and J. Bio. Opt. will contain additional and often unwanted contributions from sample areas outside the focus spot. Confocal imaging employing apertures of some kind is one way to obtain precise information on the spectral components at the focus spot without contributions from the sample above and below this point.
The article found in Biophysical Journal vol. 85, page 572-580 (2003) describes an optical probe for measuring primarily water profiles within the skin in vivo. The leg for collecting the altered light from the sample comprises an optical fiber, where coupling of the light into the fiber provides means for collecting a confocal image due to the small aperture-like diameter of the fiber. The probe comprises two lasers which can provide the incoming light and which are both focused onto the skin by a microscope objective. As the two lines for the incoming light do not use fibers, the microscope objective needs to be mounted at a fixed position. As a consequence, there is a reduced accessibility of sample spots, i.e. skin areas, which can be examined using this probing setup. Among others, diagnostics of skin abnormalities in the cervix in vivo, i.e. examination of the potential risk of cervical cancer, is excluded with this optical probe.
An optical probe for measuring optical signals in vivo, which is flexible, portable and accurately accounts for both variations in the incoming light and unwanted light signals from outside the sample focus spot, is therefore needed.